EP3240902B1 - Chitin, hydrolysate and method for the production of one or more desired products from insects by means of enzymatic hydrolysis - Google Patents

Description

The present invention relates to a process for producing at least one product of interest from insects. More particularly, the invention relates to a process for producing chitin and / or chitosan by enzymatic hydrolysis of insect cuticles. It also targets a specific chitin and a hydrolyzate.

According to the invention, by “chitin” is meant any type of chitin derivative, that is to say of polysaccharide derivative comprising N-acetyl-glucosamine units and D-glucosamine units, in particular chitin- copolymers. polypeptides (sometimes referred to as a "chitin-polypeptide composite").

Chitin is believed to be the second most synthesized polymer in the living world after cellulose. Indeed, chitin is synthesized by many species of the living world: it partly constitutes the exoskeleton of crustaceans and insects and the side wall that surrounds and protects fungi. More particularly, in insects, chitin thus constitutes 3 to 60% of their exoskeleton.

By “chitosan” is meant according to the present invention the deacetylation products of chitin. The usual limit between chitosan and chitin is determined by the degree of acetylation: a compound having a degree of acetylation less than 50% is called chitosan, beyond that, a compound having a degree of acetylation greater than 50% is named chitin.

The applications of chitin and / or chitosan are numerous: cosmetic (cosmetic composition), medical and pharmaceutical (pharmaceutical composition, treatment of burns, biomaterials, corneal dressings, surgical threads), dietetics and food, technique (filtering agent, texturizer, flocculant or adsorbent in particular for filtration and depollution of water), etc. Indeed, chitin and / or chitosan are biocompatible, biodegradable and non-toxic materials.

The traditional extraction of chitin is carried out chemically from crustaceans, cephalopods, but also, more exceptionally, fungi. This route employs large amounts of reagents (such as hydrochloric acid, sodium hydroxide, and bleaches) which have the effect of denaturing the structure of chitin as it exists naturally, for example example as present in the shell of crustaceans. In addition, most chemical reagents are harmful to humans and the environment and generate significant volumes of effluents to be treated. Finally, chitin and / or chitosan obtained from crustaceans can generate allergic reactions in sensitive people.

Another route for extracting chitin is the enzymatic route. This route is considered to be softer, thus making it possible to better preserve chitin and / or chitosan. However, the chitin obtained by this route is of a brownish color, requiring purification steps in order to obtain a recoverable powder, that is to say of white color. The existing methods therefore generally comprise one or more step (s) aimed at ridding the chitin of its impurities, such as an acid demineralization step carried out prior to the enzymatic hydrolysis and / or a chitin bleaching step. with an oxidizing agent, carried out subsequent to enzymatic hydrolysis. Unfortunately, these two chitin purification steps have the effect of altering the chemical structure of chitin.

The work of the inventors made it possible to demonstrate that it was possible to obtain a chitin that is both purer and of a structure closer to the original structure of chitin by performing a mechanical treatment step prior to hydrolysis, namely a step of pressing the cuticles of insects.

1. (i) le broyage des cuticules d'insectes,
2. (ii) le pressage des cuticules d'insectes, puis,
3. (iii) l'hydrolyse enzymatique des cuticules d'insectes par une enzyme protéolytique.

The invention therefore relates to a process for producing at least one product of interest from insects, comprising the following steps:

1. (i) crushing insect cuticles,
2. (ii) pressing the insect cuticles, then,
3. (iii) enzymatic hydrolysis of insect cuticles by a proteolytic enzyme.

By “product of interest” is meant more particularly chitin and / or chitosan and / or a hydrolyzate.

The term “hydrolyzate” denotes a product which comprises proteins, hydrolyzed proteins, peptides, amino acids and / or other compounds derived from a protein, capable of being obtained by enzymatic hydrolysis of proteins.

The product (s) of interest are obtained from insects. By "insects" is meant insects at any stage of development, such as an adult, larval or pupa stage. Preferably, the insects used in the process according to the invention are edible.

More particularly, the insects can be chosen from the group consisting of Coleoptera, Diptera, Lepidoptera, Isoptera, Orthoptera, Hymenoptera, Blattoptera, Hemyptera, Heteroptera, Ephemeroptera and Mecoptera, preferably from among beetles, Diptera, Orthoptera and Lepidoptera.

Preferably, the insects are chosen from the group consisting of Tenebrio molitor, Hermetia illucens, Galleria mellonella, Alphitobius diaperinus, Zophobas morio, Blattera fusca, Tribolium castaneum, Rhynchophorus ferrugineus, Musca domestica, Chrysomya megacephalus, Locusta migrator, and Locusta Achiephala, Locusta migrator Samia Ricini.

More preferably, the insects are chosen from the group consisting of Tenebrio molitor, Hermetia illucens, Galleria mellonella, Alphitobius diaperinus, Zophobas morio, Blattera fusca, Musca domestica, Chrysomya megacephala, Locusta migratoria, Schistocerca gregaria, Achicini domesticus, and Samia more preferably, T. molitor.

One or more species of insects can be used in the method according to the invention, preferably a single species of insect. If several species are used, two closely related species will advantageously be chosen, such as, for example, Hermetia illucens and Musca domestica.

The insects are preferably reared and not collected from the wild. For example, insects are raised on an insect farm. Raising insects on a specific farm not only controls and eliminates the risks associated with diseases carried by insects, but also limits the risks associated with the toxicity of food products derived from insects due, for example, to the presence insecticides. In addition, animal husbandry helps control the quality of insect supply and limits supply costs.

The term “insect cuticles” refers not only to the cuticles once they have been separated from the insects, but also to the cuticles including all or some of the other constituents of the insect, including the insect in its entirety. Indeed, it is possible to apply the method according to the invention to the complete insect, such as crushed insects, or else to only a part of the insects comprising the cuticles, for example the exuviae and / or the moults of insects, naturally separated and collected by a suitable process.

• l'épicuticule, qui est la couche la plus fine et la plus externe de la cuticule (inférieure à 4 µm) ; cette couche est imperméable à l'eau et comporte une couche de cire imperméabilisante, ainsi que des protéines et de la chitine, en quantité moindre ;
• l'exocuticule, qui est la couche intermédiaire de la cuticule ; elle est composée essentiellement de protéines durcies, les tannées, qui sont responsables de la rigidité de la cuticule, de chitine et éventuellement de mélanine ; et
• l'endocuticule, qui est une couche fine, flexible, constituée d'un mélange de protéines et de chitine.

The cuticle is the outer layer (or exoskeleton) secreted by the epidermis of insects. It is generally formed of three layers:

• the epicuticle, which is the thinnest and outermost layer of the cuticle (less than 4 µm); this layer is waterproof and has a waterproof wax layer, along with protein and chitin, in less quantity;
• the exocuticle, which is the middle layer of the cuticle; it is essentially composed of hardened proteins, the tanned ones, which are responsible for the rigidity of the cuticle, of chitin and possibly of melanin; and
• the endocuticle, which is a thin, flexible layer made up of a mixture of protein and chitin.

The main objective of pressing insect cuticles is to eliminate a press juice rich in fat and / or to enrich the press cake with a substrate for hydrolysis.

In the method according to the invention, the pressing of the insect cuticles makes it possible to obtain a press cake comprising an oil (or lipid) content of less than or equal to 20%, preferably less than or equal to 15%, more preferably , less than or equal to 12%, even more preferably, less than or equal to 10%.

In the present application, the ranges of values are understood to be limits included. Likewise, when "about" or "of the order of" precedes a number, it is equivalent to plus or minus 10% of the value of that number.

Furthermore, in order to enrich the press cake with a substrate for hydrolysis, pressing the insect cuticles makes it possible to obtain a press cake having a dry matter content of between 30% and 60%, preferably of between between 40% and 55%, and more preferably between 45% and 50%.

Any press system can be used to press insect cuticles, such as, for example, a single-screw or twin-screw press (Angel-type twin-screw press), a filter press (filter press of Choquenet type), a platen press, etc. These systems are well known to those skilled in the art who are able to determine the pressing conditions in order to obtain the oil and / or water contents mentioned above.

In the method according to the invention, the pressing of the insect cuticles is followed by enzymatic hydrolysis.

Preferably, the enzymatic hydrolysis is carried out by at least one proteolytic enzyme, preferably a protease. In the present application, the names or suffixes “peptidase” and “protease” are used interchangeably to designate an enzyme lysing a peptide bond of proteins. Advantageously, this is carried out for a period of 4 to 8 hours, preferably, for 4 to 5 hours, at a temperature of 40 to 60 ° C, preferably 45 to 55 ° C and at a pH between 6 and 8, preferably between 6.5 and 7.5.

The enzymatic hydrolysis can be carried out with a single protease or alternatively with a mixture of enzymes containing at least one protease, more preferably a mixture of enzymes containing several proteases, such as a mixture containing an endoprotease and an exoprotease, or a protease and a polysucrase.

Preferably, the protease is chosen from the group consisting of aminopeptidases, metallocarboxypeptidases, serine endopeptidases, cysteine endopeptidases, aspartic endopeptidases and metalloendopeptidases.

Advantageously, the enzymes can be chosen from the following: Enzyme (s) Class EC number Provider City Country Flavourzyme Aminopeptidases EC 3.4.11.1 Novozyme Bagsvaerd Denmark Fungal protease 500 EC 3.4.11.1 Bio-Cat Troy United States Kojizyme EC 3.4.11.1 Novozyme Bagsvaerd Denmark Protex P Serine endopeptidases EC 3.4.21 Genencor International BV Leiden Netherlands Chymotrypsin EC 3.4.21.1 Novozyme Bagsvaerd Denmark Protamex EC 3.4.21 Novozyme Bagsvaerd Denmark Elastase EC 3.4.21.14 Novozyme Bagsvaerd Denmark Trypsin EC 3.4.21.36 Novozyme Bagsvaerd Denmark Alkalase EC 3.4.21.4 Novozyme Bagsvaerd Denmark Papain Cysteine endopeptidases EC 3.4.22.2 Bio-Cat Troy United States Bromelaine (ananase) EC 3.4.22.32 Bio-Cat Troy United States Prolyve NP Aspartic endopeptidases EC 3.4.23 Lyven Colombelles France Pepsin EC 3.4.23.1 Sigma Aldrich Saint-Quentin-Fallavier France Neutral protease Metallo-endo-peptidase EC 3.4.24.28 Bio-Cat Troy United States Protex 50FP Endo-peptidase EC 3.4.21 Genencor International BV Leiden Netherlands Pancrealyve Exo & endo peptidase (cocktail proteases + amylases) n / A* Lyven Colombelles France Izyme BA Aspartic protease EC 3.4.23 Novozyme Bagsvaerd Denmark Sumizyme Enzymatic cocktail n / A* Takabio - Shin Nihon Aichi Japan Neutrase Zn-based endoprotease from β amyloliquefaciens EC 3.4.24 Novozyme Bagsvaerd Denmark Novozyme 37071 Protease n / A* Novozyme Bagsvaerd Denmark * na: not applicable

Advantageously, the enzyme used during the hydrolysis is an aspartic endopeptidases, such as Prolyve NP. This type of enzyme makes it possible to obtain very good results in terms of the purity of the chitin obtained, in particular when this type of enzyme is applied to the hydrolysis of a press cake obtained from beetles and more particularly by T. molitor.

The enzyme or the mixture of enzymes is introduced in an amount ranging from 0.2 to 10% by weight of estimated dry matter, preferably from 0.4 to 8% by weight and more preferably from 0.5 to 2%. The term “estimated dry matter weight” more particularly refers to the dry matter weight of insects or insect part (s), as can be estimated when entering the enzymatic hydrolysis step. .

In terms of enzymatic activity, the quantity of enzyme or mixture of enzymes introduced is equivalent to an activity of between 2000 and 5000 SAPU (“ Spectrophotometric Acid Protease Unit ”, described in Example 5 below), of preferably between 3000 and 4000 SAPU, per 100 g wet weight, with a humidity of 30 to 70%, of the substrate to be transformed, that is to say of insect material or hydrated insect part (s).

Advantageously, the enzymatic hydrolysis step is carried out in the presence of water, such as fresh water. The amount of water used during the enzymatic hydrolysis is determined as follows: the ratio of the volume of water in mL to the weight in g of insect is preferably between 0.3 and 10, more preferably between 0.5 and 5, even more preferably between 0.7 and 3, even more preferably of the order of 1. It will be noted that this ratio also corresponds to the ratio of the weight of water to the weight of the insect, the density of water being 1.0 g / mL under normal conditions of temperature and pressure.

The process according to the invention makes it possible to obtain a chitin having a degree of purity (or gravimetric purity) of between 40 and 90%, preferably between 50 and 90%, more preferably between 60 and 85%, and even more. preferably of the order of 80% (see Example 2 and Figure 2 ).

In addition, the process according to the invention makes it possible to obtain a hydrolyzate exhibiting a certain number of advantageous properties, in particular in terms of digestibility, of lipid and / or protein content, of protein size or of acid composition. amines.

The method according to the invention comprises a grinding step prior to the pressing step.

The purpose of this grinding step is to reduce the cuticles and / or the insects into particles in order to facilitate the access of the enzymes to the substrate during the enzymatic hydrolysis. This step also makes it possible, when it is followed by a pressing step, to facilitate the elimination of the press juice and the isolation of the solid material.

This grinding step also makes it possible to improve the distribution of the lipids obtained from the insect between the press cake and the press juice obtained from the pressing. Indeed, as illustrated in Figure 4 , the press cake resulting from a process comprising both grinding and pressing has a reduced lipid content in comparison with a press cake from a process comprising only pressing.

More particularly, in the process according to the invention, the grinding and pressing of the insect cuticles makes it possible to obtain a press cake comprising a lipid (or oil) content of less than or equal to 15%, preferably less than or equal at 12%, even more preferably, less than or equal to 10%.

The grinding can advantageously be carried out with a mixer mill, such as a mixer mill with knives.

Preferably, after grinding, the size of the insect particles is less than 1 cm (largest particle size observable using a microscope), preferably less than 0.5 cm, more preferably again, a size of between 300 μm and 0.5 cm, preferably 500 μm and 0.5 cm and even more preferably between 500 μm and 1 mm.

In order to facilitate the grinding, a quantity of water can be added. This quantity of water is determined as follows: the ratio of the volume of water in mL to the weight in g of insect is preferably between 0.3 and 10, more preferably between 0.5 and 5, again more preferably between 0.7 and 3, even more preferably of the order of 1.

Advantageously, the method according to the invention also comprises a step of slaughtering the insects prior to the step of pressing and / or grinding.

This slaughter step can be carried out by conventional methods of rearing cold-blooded and / or small animals (crustaceans, fish, snails, etc.), such as cold (freezing), heat ( scalding), oxygen deprivation, etc. Advantageously, the step of slaughtering the insects is carried out by scalding. Scalding not only kills insects, but also lowers the microbial load (reduced risk of spoilage and health) and inactivates internal enzymes in insects that can trigger autolysis, and thus rapid browning of the insects. these. This scalding is carried out in such a way as to cause death as quickly as possible, while respecting animal welfare, and according to scientific recommendations.

Alternatively, slaughter can be done by blanching. Bleaching has the same advantages mentioned above as scalding.

Advantageously, the insects are slaughtered, for example by scalding or bleaching, then crushed before being pressed.

Preferably, the scalding step is carried out in water, such as fresh water, at a temperature of 95 to 105 ° C, preferably of the order of 100 ° C and for a period of 2 to 20 min, preferably 5 to 15 min.

The amount of water introduced at this scalding step is determined as follows: the ratio of the volume of water in mL to the weight in g of insect is preferably between 0.3 and 10, more preferably between 0.5 and 5, even more preferably between 0.7 and 3, even more preferably of the order of 1.

Alternatively, the bleaching step is carried out with steam and / or with water at a temperature between 80 ° C and 130 ° C, preferably between 90 ° C and 120 ° C.

The method according to the invention may further comprise a step of treating the cuticles of insects with an oxidizing agent prior to enzymatic hydrolysis.

Preferably, in the process according to the invention, the oxidizing agent used during the treatment of the cuticles is chosen from the group consisting of hydrogen peroxide, potassium permanganate, ozone and sodium hypochlorite , more preferably still, hydrogen peroxide.

Advantageously, when the oxidizing agent is hydrogen peroxide, the amount of this agent introduced for the treatment of insect cuticles is such that the hydrogen peroxide content is between 1 and 33% by weight on the weight. total of insects, preferably between 2 and 12% by weight on the total weight of insects, preferably of the order of 6% by weight.

Preferably, the treatment of insect cuticles with the oxidizing agent is carried out in the presence of water, such as fresh water. Advantageously, the amount of water used during the treatment of the cuticles is determined as follows: the ratio of the volume of water in mL to the weight in g of insect is preferably between 0.3 and 10, more preferably between 0.5 and 5, even more preferably between 0.7 and 3, even more preferably of the order of 1.

• concomitamment avec l'ébouillantage et/ou après l'étape d'ébouillantage, plus préférentiellement concomitamment avec l'ébouillantage. Alternativement, concomitamment avec le blanchiment et/ou après l'étape de blanchiment, plus préférentiellement concomitamment avec le blanchiment. Plus particulièrement, lorsque le traitement des cuticules d'insectes est effectué pendant l'ébouillantage ou le blanchiment, l'agent oxydant peut être avantageusement ajouté à l'eau utilisée pour ébouillanter les insectes.
• avant, concomitamment et/ou après le broyage. Plus particulièrement, lorsque le traitement des cuticules d'insectes est effectué pendant le broyage, l'agent oxydant peut être avantageusement ajouté à l'eau utilisée pour le broyage.
• avant et/ou concomitamment avec le pressage.
• lors d'une étape spécifique de traitement des cuticules d'insectes.

Treatment of insect cuticles with the oxidizing agent can be carried out during one or more of the following steps:

• concomitantly with scalding and / or after the scalding step, more preferably concomitantly with scalding. Alternatively, concomitantly with the bleaching and / or after the bleaching step, more preferably concomitantly with the bleaching. More particularly, when the treatment of insect cuticles is carried out during scalding or bleaching, the oxidizing agent can be advantageously added to the water used for scalding the insects.
• before, concomitantly and / or after grinding. More particularly, when the treatment of insect cuticles is carried out during grinding, the oxidizing agent can be advantageously added to the water used for the grinding.
• before and / or concomitantly with pressing.
• during a specific stage of treatment of insect cuticles.

Advantageously, the enzymatic hydrolysis can be followed by a thermal inactivation step aimed at inactivating the enzyme or the mixture of enzymes used during the enzymatic hydrolysis.

At the end of a process according to the invention, the chitin can be recovered by pressing or centrifuging the reaction medium for the enzymatic hydrolysis. At this stage, a chitin co-product is also recovered, namely a hydrolyzate.

A preferred embodiment of a method according to the invention is more detailed below.

In particular, this preferred embodiment describes various advantageous steps for a process according to the invention, such as steps of gentle purification of the chitin: a second pressing, possible washing, filtration and drying.

Finally, since chitin is generally sold in powder form, a second grinding can also be carried out. This can also be carried out in order to promote the deacetylation reaction, which makes it possible to prepare chitosan from chitin. The conditions of the deacetylation reaction are more fully described in step 10 of the preferred embodiment detailed below.

1. a) l'abattage des insectes,
2. b) le broyage des insectes,
3. c) le pressage des insectes,
4. d) l'hydrolyse enzymatique des cuticules d'insectes par une enzyme protéolytique,
5. e) la récupération de la chitine,

les cuticules d'insectes pouvant optionnellement être traitées avec un agent oxydant avant l'étape d).A particularly advantageous process for producing chitin from insect cuticles comprises the following steps:

1. (a) the killing of insects,
2. b) crushing insects,
3. c) pressing of insects,
4. d) enzymatic hydrolysis of insect cuticles by a proteolytic enzyme,
5. e) recovery of chitin,

the insect cuticles can optionally be treated with an oxidizing agent before step d).

The preferred embodiments of the various steps a) to e) as well as the treatment with the oxidizing agent are as indicated above or in the corresponding step in the preferred embodiment below.

The invention also relates to a chitin, such as a chitin obtainable by a process according to the invention. Thanks to the mild conditions employed in the process according to the invention, this chitin has a structure close to chitin as present in the natural state in the cuticle of the insect while having a high degree of purity, such as degree of purity between 40 and 90%, preferably between 50 and 90%, more preferably between 60 and 85%, and even more preferably of the order of 80%.

• un taux de cendre inférieur à 4%, préférentiellement inférieur à 3,5%, plus préférentiellement inférieur ou égal à 2,5% (en particulier lorsque la chitine est préparée à partir d'insectes végétariens), plus préférentiellement inférieur à 2%, et encore plus préférentiellement inférieur ou égal à 1,5% (en particulier lorsque la chitine est préparée à partir de T. molitor), et encore plus préférentiellement inférieur à 1% en poids sur le poids total de matière sèche,
• degré de pureté (ou pureté gravimétrique) compris entre 40 et 90%, préférentiellement entre 50 et 90%, plus préférentiellement compris entre 60 et 85%, et encore plus préférentiellement de l'ordre de 80%,
• une abondance fonctionnelle en surface présentant un ratio (C-O)/(C-H) compris entre 0,30 et 0,56, préférentiellement, entre 0,31 et 0,53,
• Taux de lipides ≤14% en poids total sur le poids total de matière sèche,
• Taux de lipides ≤5% lorsque la chitine est préparée à partir d'insectes non volants,
• Total des acides aminés ≤45%,
• Total des acides aminés ≤16% lorsque la chitine est préparée à partir d'insectes volants,
• Taux d'abondance relative de au moins 3 quelconque acides aminés parmi ALA, GLY, LEU, PRO, SER, TYR, VAL ≤10%,
• Taux d'abondance relative de LEU, PRO, VAL ≤10% lorsque la chitine est préparée à partir de T. molitor,
• Taux d'abondance relative de ALA ≤12% lorsque la chitine est préparée à partir de T. molitor,
• Pureté colorimétrique ≥40%,
• Pureté colorimétrique ≥50% lorsque la chitine est préparée avec l'enzyme prolyve NP lors de l'hydrolyse enzymatique,
• Pureté par différence ≥45%, préférentiellement ≥49%,
• Pureté par différence ≥52% lorsque la chitine est préparée à partir de T. molitor,
• Pureté par différence ≥70% lorsque la chitine est préparée à partir d'insectes volants,
• Degré d'acétylation ≥70% et degré de cristallinité ≤0,61,
• Degré d'acétylation ≥70% et degré de cristallinité ≥0,42.

The chitin capable of being obtained by a process according to the invention exhibits at least any one of the following advantageous properties:

• an ash content of less than 4%, preferably less than 3.5%, more preferably less than or equal to 2.5% (in particular when the chitin is prepared from vegetarian insects), more preferably less than 2%, and even more preferably less than or equal to 1.5% (in particular when the chitin is prepared from T. molitor ), and even more preferably less than 1% by weight on the total weight of dry matter,
• degree of purity (or gravimetric purity) of between 40 and 90%, preferably between 50 and 90%, more preferably between 60 and 85%, and even more preferably of the order of 80%,
• a functional surface abundance exhibiting a (CO) / (CH) ratio of between 0.30 and 0.56, preferably between 0.31 and 0.53,
• Lipid content ≤14% by total weight on the total weight of dry matter,
• Lipid level ≤5% when chitin is prepared from non-flying insects,
• Total Amino Acids ≤45%,
• Total Amino Acids ≤16% when chitin is prepared from flying insects,
• Relative abundance rate of at least 3 any amino acids among ALA, GLY, LEU, PRO, SER, TYR, VAL ≤10%,
• Relative abundance rate of LEU, PRO, VAL ≤10% when chitin is prepared from T. molitor,
• Relative abundance rate of ALA ≤12% when chitin is prepared from T. molitor,
• Colorimetric purity ≥40%,
• Colorimetric purity ≥50% when chitin is prepared with the enzyme prolyve NP during enzymatic hydrolysis,
• Purity by difference ≥45%, preferably ≥49%,
• Purity by difference ≥52% when chitin is prepared from T. molitor,
• Purity by difference ≥70% when chitin is prepared from flying insects,
• Acetylation degree ≥70% and crystallinity degree ≤0.61,
• Degree of acetylation ≥70% and degree of crystallinity ≥0.42.

The chitin according to the invention comprises an amino acid level less than or equal to 45% by weight on the total weight of dry matter, an ash level less than or equal to 2.5% by weight on the total weight of dry matter , a purity by difference greater than or equal to 45%, a degree of acetylation greater than or equal to 70% and a degree of crystallinity greater than or equal to 0.42.

Preferably, the chitin exhibits all of the above properties.

All the units and methods of measurement of the characteristics indicated above are described in the Examples and, more particularly, in Example 5.

1. a) l'abattage des insectes,
2. b) le broyage des insectes,
3. c) le pressage des insectes,
4. d) l'hydrolyse enzymatique des cuticules d'insectes par une enzyme protéolytique,
5. e) la récupération de l'hydrolysat,

les cuticules d'insectes pouvant optionnellement être traitées avec un agent oxydant avant l'étape d).A particularly advantageous process for producing a hydrolyzate from insects comprises the following steps:

1. (a) the killing of insects,
2. b) crushing insects,
3. c) pressing of insects,
4. d) enzymatic hydrolysis of insect cuticles by a proteolytic enzyme,
5. e) recovery of the hydrolyzate,

the insect cuticles can optionally be treated with an oxidizing agent before step d).

The preferred embodiments of the various steps a) to e) as well as the treatment with the oxidizing agent are as indicated above or in the corresponding step in the preferred embodiment below.

The invention also relates to a hydrolyzate, such as a hydrolyzate obtainable by a process according to the invention.

• un taux de cendre inférieur à 4%, préférentiellement inférieur à 3% en poids (en particulier lorsque l'hydrolysat est préparé à partir d'insectes végétariens) sur le poids total de matière sèche,
• une excellente digestibilité, en particulier une digestibilité pepsique, supérieure à 95%, préférentiellement supérieure ou égale à 96% et plus préférentiellement supérieure ou égale à 98%, et plus particulièrement supérieure ou égale à 99%, la digestibilité pepsique ayant été mesurée selon une méthode conforme à la directive 72/188/EEC ;
• une teneur en protéines/peptides supérieure ou égale à 70%, préférentiellement supérieure ou égale à 71%, et plus préférentiellement supérieure ou égale à 75%, et plus préférentiellement supérieure ou égale à 80% (en particulier, lorsque l'hydrolysat est préparé à partir d'insectes non volant) ;

• une teneur en protéines hydrosolubles présentant une taille supérieure à 12400 g/mol, inférieure à 20%, de préférence, inférieure à 18% en poids sur le poids total de protéines hydrosolubles ;
• une teneur en lipides inférieure à 14%, préférentiellement inférieure à 10%, plus préférentiellement inférieure ou égale à 5% (en particulier lorsque l'hydrolysat est préparé à partir d'insectes non volants), plus préférentiellement inférieure ou égale à 2%, et encore plus préférentiellement inférieure ou égale à 1% ;
• une composition dans laquelle les acides aminés les plus abondants sont la proline, l'alanine, la leucine, l'acide aspartique, la glutamine et/ou la valine. Par « plus abondant », on entend une quantité relative de l'acide aminé dans la composition supérieure à 5%, préférentiellement supérieure à 7% en poids sur le poids total d'acides aminés. La forte teneur en proline s'avère particulièrement intéressante ;
• Taux d'abondance relative de au moins 5 quelconques acides aminés choisis parmi ASP, GLU, ALA, GLY, LEU, PRO, TYR, VAL, LYS ≥6% ;
• Taux d'abondance relative de au moins 3 quelconques acides aminés choisis parmi ASP, GLU, ALA, LEU, PRO, TYR, VAL ≥8%.

The hydrolyzate obtainable by a process according to the invention exhibits at least any one of the following advantageous properties:

• an ash content of less than 4%, preferably less than 3% by weight (in particular when the hydrolyzate is prepared from vegetarian insects) on the total weight of dry matter,
• excellent digestibility, in particular a pepsi digestibility, greater than 95%, preferably greater than or equal to 96% and more preferably greater than or equal to 98%, and more particularly greater than or equal to 99%, the pepsi digestibility having been measured according to a method in accordance with Directive 72/188 / EEC;
• a protein / peptide content greater than or equal to 70%, preferably greater than or equal to 71%, and more preferably greater than or equal to 75%, and more preferably greater than or equal to 80% (in particular, when the hydrolyzate is prepared from non-flying insects);

• a water-soluble protein content having a size greater than 12400 g / mol, less than 20%, preferably less than 18% by weight on the total weight of water-soluble proteins;
• a lipid content of less than 14%, preferably less than 10%, more preferably less than or equal to 5% (in particular when the hydrolyzate is prepared from non-flying insects), more preferably less than or equal to 2%, and even more preferably less than or equal to 1%;
• a composition in which the most abundant amino acids are proline, alanine, leucine, aspartic acid, glutamine and / or valine. By “more abundant” is meant a relative amount of amino acid in the composition greater than 5%, preferably greater than 7% by weight on the total weight of amino acids. The high proline content is particularly interesting;
• Relative abundance rate of at least any 5 amino acids selected from ASP, GLU, ALA, GLY, LEU, PRO, TYR, VAL, LYS ≥6%;
• Relative abundance rate of at least any 3 amino acids selected from ASP, GLU, ALA, LEU, PRO, TYR, VAL ≥8%.

More particularly, the term “water-soluble proteins” means, among proteins (or crude proteins), those which are soluble in an aqueous solution whose pH is between 6 and 8, advantageously between 7.2 and 7.6.

Preferably, the aqueous solution is a buffer solution the pH of which is between 6 and 8, advantageously between 7.2 and 7.6.

Preferably, the buffer solution is an NaCl phosphate solution, the pH of which is equal to 7.4 ± 0.2.

The hydrolyzate according to the invention comprises at least 40% of proteins by weight on the total weight of dry matter, an ash content of less than 3% by weight on the total weight of dry matter, and a rate of water-soluble proteins having a size greater than 12400 g / mol less than 50%, preferably less than 43%.

The term “vegetarian insect” is intended to mean an insect which does not have animal proteins in its usual diet. By way of example of a vegetarian insect, mention may be made of coleoptera, lepidoptera or orthoptera.

By "flying insect" we mean an insect which is able to fly at age adult, unlike a so-called "non-flying" insect. By way of example of a flying insect, there may be mentioned the Lepidoptera or the Diptera. By way of example of a non-flying insect, mention may be made of coleoptera or orthoptera.

All the units and methods of measurement of the characteristics indicated above are described in the Examples and, more particularly, in Example 5.

Preferably, the hydrolyzate has all of the above properties.

It will be noted in particular that the hydrolyzate can be distinguished from any other type of hydrolyzate by its glucosamine content, and / or one of its derivatives (preferably N- acetyl -glucosamine), more particularly a content greater than or also at 0.01%, preferably greater than or equal to 0.08% by weight on the total weight of dry matter of the hydrolyzate.

In the present application, any reference to a regulation or to a directive concerns said regulation or said directive as in force on the date of filing of the present application.

The hydrolyzate can advantageously be supplemented with additives to balance its nutritional profile in order to be suitable for different types of animals.

The hydrolyzate can advantageously be concentrated and then dried to obtain a dried hydrolyzate. Alternatively, the hydrolyzate can be in liquid form. These hydrolysates can be used as a food or food ingredient in particular for animals, or, alternatively, they can be processed, for example, to isolate amino acids.

1. a) l'abattage des insectes,
2. b) le broyage des insectes,
3. c) le pressage des insectes,
4. d) l'hydrolyse enzymatique des cuticules d'insectes par une protéase,
5. e) la récupération de la chitine,
6. f) la désacétylation de la chitine récupérée,
7. g) la récupération du chitosan,

les cuticules d'insectes pouvant optionnellement être traitées avec un agent oxydant avant l'étape d).A particularly advantageous process for producing chitosan from insect cuticles comprises the following steps:

1. (a) the killing of insects,
2. b) crushing insects,
3. c) pressing of insects,
4. d) enzymatic hydrolysis of insect cuticles by a protease,
5. e) recovery of chitin,
6. f) deacetylation of the recovered chitin,
7. g) recovery of chitosan,

the insect cuticles can optionally be treated with an oxidizing agent before step d).

The preferred embodiments of the various steps a) to g) as well as the treatment with the oxidizing agent are as indicated above or in the corresponding step in the preferred embodiment below.

The invention also relates to a chitosan obtainable by a process according to the invention.

• dans des compositions cosmétiques, pharmaceutiques, nutraceutiques ou diététiques,
• comme biomatériaux pour traiter des brûlures en tant que seconde peau, pour réaliser des pansements cornéens ou des fils chirurgicaux,
• comme agent filtrant, texturant, floculant et/ou adsorbant notamment pour la filtration et dépollution de l'eau.

The chitin and / or the chitosan capable (s) of being obtained by a process according to the invention can advantageously be used (s) in various applications:

• in cosmetic, pharmaceutical, nutraceutical or dietetic compositions,
• as biomaterials for treating burns as a second skin, for making corneal dressings or surgical threads,
• as a filtering, texturizing, flocculant and / or adsorbent agent, in particular for the filtration and depollution of water.

According to a preferred embodiment of the invention, the method comprises the following steps, described schematically in Figure 1 . It will be noted that certain steps are indicated as optional in this preferred embodiment.

• Step 1: slaughter of insects

This slaughter step 1 makes it possible to kill the insects while lowering the microbial load (reduction of the risk of spoilage and health) and by inactivating the internal enzymes of the insects which can trigger autolysis, and thus a rapid browning of them. .

The slaughter can be done by scalding.

The insects, preferably larvae, are thus scalded with water for 2 to 20 min, preferably 5 to 15 min. Preferably, the water is at a temperature of between 95 to 105 ° C, preferably 100 ° C.

The amount of water introduced in this scalding step 1 is determined as follows: the ratio of the volume of water in mL to the weight in g of insect is preferably between 0.3 and 10, more preferably between 0.5 and 5, even more preferably between 0.7 and 3, even more preferably of the order of 1.

Alternatively, slaughter can be done by bleaching. Preferably, the insects are bleached with steam (nozzles or steam bed) at a temperature between 80 and 130 ° C, preferably between 90 and 120 ° C, more preferably between 95 and 105 ° C, even more preferably 98 ° C or else with water at a temperature between 95 and 105 ° C, preferably 100 ° C (by spray nozzles) or in mixed mode (water + steam) at a temperature between 80 and 130 ° C, preferably between 90 and 120 ° C, more preferably between 95 and 105 ° C. The residence time in the bleaching chamber is between 1 and 15 minutes, preferably between 3 and 7 min.

During this step, it is also possible to treat the insect cuticles by using a scalding or bleaching water comprising the oxidizing agent according to the procedures indicated in the intermediate step below.

• Intermediate step (optional) : treatment of the cuticles with the oxidizing agent

It is possible to introduce into the process a specific step of treatment of the cuticles with the oxidizing agent. Advantageously, this intermediate cuticle treatment step is carried out between step 1 of slaughtering and step 2 of grinding.

This intermediate step is preferably carried out with an oxidizing agent chosen from the group consisting of hydrogen peroxide (H ₂ O ₂ ), potassium permanganate (KMnO ₄ ), ozone (O ₃ ) and hypochlorite sodium (NaClO), more preferably hydrogen peroxide.

According to a first embodiment, at the end of step 1, when this is done by scalding, the oxidizing agent is introduced directly into the scalding tank, after optional cooling of the scalding water. at a temperature of the order of 40 to 60 ° C, preferably of the order of 50 ° C.

The hydrogen peroxide as marketed is usually in the form of an aqueous solution, for example a solution at 30% by weight on the total weight of water.

The amount of hydrogen peroxide introduced for the treatment is such that the hydrogen peroxide content is between 1 to 33% by weight on the total weight of insects, preferably 2 to 12% by weight on the total weight of insects, preferably of the order of 6% by weight.

According to a second embodiment, the insects are removed from the scalding tank, sieved and reintroduced into a tank.

The hydrogen peroxide is then introduced into the tank in the form of a dilute aqueous solution, the hydrogen peroxide content then being between 1 and 33% by weight on the weight of water, preferably 2 to 12 % by weight on the weight of water, preferably of the order of 6% by weight.

• Step 2 : grinding

The insects are removed from the scalding or treatment tank or from the blanching chamber, they are then sieved, and placed in a crusher, such as a knife mill, to reduce the insects to particles.

In order to facilitate the grinding, a quantity of water can be added. This quantity of water is similar to that introduced during scalding stage 1: the ratio of the volume of water in mL on the weight in g of insect is preferably between 0.3 and 10, more preferably between 0.5 and 5, even more preferably between 0.7 and 3, even more preferably of the order of 1. It is also possible to keep the scalding water and / or the water from the intermediate step to perform this step. This water is likely to contain the oxidant. In this case, the treatment of the cuticles can take place during stage 1 of scalding and stage 2 of grinding or during the intermediate stage of treatment of the cuticles and during the grinding stage.

Preferably, after grinding, the size of the insect particles is less than 1 cm (largest particle size observable using a microscope), preferably less than 0.5 cm. Preferably, the size of the particles is between 500 μm and 0.5 cm and even more preferably between 500 μm and 1 mm.

It is not necessary to reduce the particle size excessively, for example to a size less than 250 µm.

This grinding step 2 promotes the access of enzymes to their substrate.

During this step, it is possible to introduce the oxidizing agent into the grinder in order to treat the cuticles according to the methods indicated in the intermediate step above.

When the treatment of the cuticles is not carried out concomitantly with the grinding, it is possible to add, during this step, antioxidant additives commonly used for the preservation and stability of the product.

• Step 3 : pressing the insect cuticles

The wet paste resulting from grinding step 2 is then placed in a press according to an operating method which makes it possible to press and separate a juice comprising both an oily fraction and a protein fraction.

Preferably, the pressing step makes it possible to obtain a press cake comprising an oil content of less than or equal to 20%, preferably less than or equal to 15%, more preferably less than or equal to 12%, even more preferably , less than or equal to 10%.

Likewise, the pressing step makes it possible to obtain a press cake having a dry matter content of between 30% and 60%, preferably between 40% and 55%, and more preferably between 45% and 50%. .

Any press system can be used to perform the pressing step, such as, for example, a single-screw or twin-screw press (Angel-type twin-screw press), a filter press (Choquenet type filter press), a plate press, etc. These systems are well known to those skilled in the art who are able to determine the pressing conditions in order to obtain the oil and / or water contents mentioned above.

If the wet pulp resulting from step 2 of grinding was obtained with water comprising the oxidant, it may be advantageous to remove at least part of this oxidant before step 3 of pressing.

This pressing step 3 can optionally be carried out before the grinding step 2 from the scalded insects. However, it is advantageous to perform step 3 of pressing after step 2 of grinding.

This pressing step 3 therefore makes it possible to obtain a press juice and a press cake.

• Step 4: enzymatic hydrolysis

The wet paste resulting from step 2 of grinding or the press cake resulting from step 3 of pressing is placed in a hydrolysis tank with water.

Optionally, and as will be described below, the protein fraction resulting from separation step 12 can be reintroduced into this enzymatic hydrolysis step 4, by mixing it with the press cake.

Optionally and in the case where the boiling water does not contain an oxidant, the boiling water can be recovered and reintroduced during the hydrolysis step. Indeed, this water contains insect fractions solubilized by the action of this scalding and the use of this during hydrolysis makes it possible to reduce losses. Optionally, this water resulting from the boiling can be degreased, some waxes possibly having dissolved in the water.

The amount of water introduced in this enzymatic hydrolysis step 4 is similar to that introduced during scalding step 1: the ratio of the volume of water in mL to the weight in g of insect is preferably included between 0.3 and 10, more preferably between 0.5 and 5, even more preferably between 0.7 and 3, even more preferably of the order of 1.

The enzymatic hydrolysis is carried out with a protease, such as a commercial protease for 4 to 8 hours, more particularly for 4 to 5 hours, at a pH of 6 to 8, more particularly of 6.5 to 7.5, at a temperature from 40 to 60 ° C, more particularly from 45 to 55 ° C.

The quantity of enzymes introduced during the hydrolysis step is less than 10% by weight on the total weight of estimated dry matter entering hydrolysis, preferably less than 6%, more preferably of the order of 2%.

Proteolytic hydrolysis results in the production of a soluble phase containing peptides, glucosamines and oligochitins and of a solid residue formed of chitin, mainly chitin-polypeptide copolymer.

• Step 5 : thermal inactivation

In order to stop the activity of the enzymes of the reaction and to stabilize the soluble phase of the hydrolysis, thermal inactivation is carried out by heating this juice between 80 and 105 ° for 10 to 25 minutes, preferably 15 to 20 minutes. According to one procedure, this thermal inactivation step 5 is carried out according to the usual sterilization techniques in the food industry. According to another procedure, the enzymatic inactivation is carried out by heating under IR or UV radiation, or by microwave.

• Step 6 : pressing

The solid residue, mainly composed of chitin, is recovered and then pressed by a press in order to wring out this residue as much as possible in order to reinject this pressate into the soluble phase. The pressed residue thus formed is composed essentially of chitin, mainly in the form of chitin-polypeptide copolymer.

• Steps (optional) 7 and 8 : washing and drying

The solid residue is then washed, filtered, washed again and then dried by conventional technologies known to those skilled in the art.

Advantageously, the drying system is designed to protect the structure of the chitin-polypeptide copolymer: the hydrometry, ventilation and the composition of the air are controlled. Advantageously, the drying can be carried out in a ventilated oven at a temperature of 60 to 80 ° C, preferably of the order of 70 ° C.

Optionally, these steps can include a terminal defatting step: the solid residue is treated with HCl in order to remove the last lipid residues, in particular the cuticular waxes.

Steps 9 to 11 which follow aim at transforming chitin into chitosan and are therefore implemented only when the desired product is chitosan.

• Step 9 (optional) : grinding

The dried solid residue, mainly comprising chitin, is then ground, for example in a knife mill.

The production of chitosan from chitin, by the deacetylation reaction, depends largely on the size of the chitin particles. Thus a very fine grinding of the solid residue dried before deacetylation makes it possible to significantly increase the yields and the speed of the deacetylation reaction, as illustrated in Table 1 below: <b> Table 1 </b>: <b> Efficiency of deacetylation according to the prior grinding of the chitin </b> Grinding 30 s Grinding 45 s Grinding 60 s Grinding 120 s 50% of particles <174 µm <117 µm <95 µm <67 µm 90% of particles <310 µm <244 µm <157 µm <159 µm DA * 99% 90% 85% 80% * Measurement of the Degree of Acetylation DA

The conditions of the deacetylation carried out in the test reported in Table 1 are as follows: 4h of reaction, 100 ° C, NaOH in aqueous solution at 30% by volume, in an estimated chitin ratio: NaOH solution equal to 1: 20.

Consequently, the solid residue is preferably ground to a particle size of less than 200 μm, more preferably, less than 160 μm.

• Step 10 : deacetylation

The solid residue, optionally crushed in step 9, is then placed in a reactor to which a concentrated sodium hydroxide solution is added for 4 to 24 hours, and preferably 6 to 18 hours. The sodium hydroxide in aqueous solution at a content ranging from 30% to 40% is added according to a weight ratio in g of ground chitin / volume in mL of sodium hydroxide in aqueous solution of between 1: 50 to 1: 10 , preferably of the order of 1:20. The tank is then heated, the deacetylation temperature being between 80 and 150 ° C, preferably between 90 and 120 ° C and more preferably at 100 ° C.

Powdered chitosan is thus obtained.

The chitosan can then undergo any operation known to those skilled in the art making it possible to functionalize it, in particular by adding radicals (carboxylation, hydroxylation, etc.)

• Step 11 (optional) : drying

The chitosan powder is then dried between 30 and 80 ° C, preferably between 50 and 70 ° C and preferably at about 60 ° C, in order to obtain a powder having a dry matter content greater than 85%, more particularly greater than 90%.

The steps which follow aim to recover an oily fraction and a protein fraction from the juice obtained in pressing step 3 and are therefore carried out only when such recovery is desired.

• Step 12 : separation

The press juice undergoes one or more separation steps in order to separate the oily fraction (insect oils) from the protein fraction (insects hemolymph proteins). These steps can be carried out by any oil separation technology well known to those skilled in the art, such as centrifugation, decantation, separation by reverse osmosis, ultrafiltration, supercritical CO ₂ , etc. or a combination of several of these technologies.

The separation of the oily fraction can be complex, given the presence of oils of very different compositions in insects, the fatty acids being able to present short chains (C2-C5) as well as very long chains (for example, for waxes:> C25). The rise in temperature above the melting point of these oils (approximately 38 ° C.) during centrifugation makes it possible to dissolve this cream and to facilitate the separation of the oily fraction from the rest of the juice. The centrifugate then goes into decantation according to an operating mode (decanter or tricanter type), in order to separate the oils and proteins as well as possible.

These steps thus make it possible to obtain an oily fraction.

The protein fraction, once separated from the oily fraction, can be mixed with the press cake resulting from pressing step 3 just before hydrolysis step 4. Indeed, often more than 20% of the proteins are lost in the juice during step 3 of pressing, hence the advantage of recovering this fraction and subjecting it to the hydrolysis step.

• Step 13 (optional) : concentration

According to one procedure, the concentration is carried out by vacuum evaporation of the aqueous part. The concentrate has a dry extract greater than 10%, preferably greater than 20%. This operation facilitates drying and additives commonly used for the preservation and stability of the product can be added at this stage.

• Step 14 (optional) : drying

The concentrate is finally dried using technologies known to those skilled in the art, such as, for example, by spraying / atomization (“spray-drying”), which makes it possible to obtain an extract, that is to say a powder. dry concentrate rich in peptides and glucosamines, the glucosamines being obtained in particular from partial hydrolysis of chitin by H ₂ O ₂ (mainly).

• La Figure 1 est un schéma d'un mode de réalisation préférentiel du procédé selon l'invention,
• La Figure 2 un diagramme comparant le degré de pureté de la chitine obtenue par un procédé enzymatique comportant une ou plusieurs étapes préalables de broyage(s) et pressage,
• La Figure 3 est un diagramme comparant le taux de lipides mesuré dans différentes fractions du produit intermédiaire dont la chitine a été extraite,
• La Figure 4 illustre la répartition des lipides dans le jus et le gâteau de presse obtenus par un procédé enzymatique comportant des étapes préalables de broyage et de pressage ou une étape préalable de pressage,
• La Figure 5 est un diagramme illustrant la répartition des protéines/peptides selon leurs masses molaires (en %) dans un hydrolysat selon l'invention et dans le jus de presse,
• La Figure 6 est un diagramme illustrant la répartition relative des acides aminés constitutifs d'un l'hydrolysat selon l'invention,
• La Figure 7 est un diagramme illustrant l'abondance relative des acides aminés dans la chitine ;
• La Figure 8 : Effet du pressage sur le taux de cendres dans l'hydrolysat - différents insectes,
• La Figure 9 : Effet du pressage sur le taux de cendres dans l'hydrolysat - différentes enzymes,
• La Figure 10 : Taux de cendres dans l'hydrolysat avec le procédé broyage + pressage - différents insectes,
• La Figure 11 : Abondance relative de protéines de grande taille en fonction de différents procédés et différents insectes pour une hydrolyse effectués avec la Prolyve,
• La Figure 12 : Abondance relative de protéines de grande taille en fonction de différents procédés et différentes enzymes appliquées à T. molitor,
• La Figure 13 : Effet du pressage sur la teneur en protéines de l'hydrolysat - différentes enzymes,
• La Figure 14 : Effet du pressage sur la teneur en protéines de l'hydrolysat - différents insectes,
• La Figure 15 : Effet du pressage sur la teneur en lipides de l'hydrolysat - différentes enzymes,
• La Figure 16 : Effet du pressage sur la teneur en lipides de l'hydrolysat - différents insectes,
• La Figure 17 : Digestibilité pepsique des hydrolysats obtenus - provisoire,
• La Figure 18: TM + prolyve : abondance relative des acides aminés de l'hydrolysat,
• La Figure 19 : TM + sumizyme : abondance relative des acides aminés de l'hydrolysat,
• La Figure 20 : TM + novozyme : abondance relative des acides aminés de l'hydrolysat,
• La Figure 21 : TM + neutrase : abondance relative des acides aminés de l'hydrolysat,
• La Figure 22 : HI + prolyve : abondance relative des acides aminés de l'hydrolysat,
• La Figure 23 : GM + prolyve : abondance relative des acides aminés de l'hydrolysat,
• La Figure 24 : AD + prolyve : abondance relative des acides aminés de l'hydrolysat,
• La Figure 25 : Effet du pressage sur le taux de cendres dans la chitine - différentes enzymes,
• La Figure 26 : Taux de cendres dans la chitine de différents insectes,
• La Figure 27 : Teneur en lipides dans la chitine,
• La Figure 28 : Taux d'acides aminés dans la chitine,
• La Figure 29 : TM + prolyve : abondance relative des acides aminés de la chitine,
• La Figure 30 : TM + sumizyme : abondance relative des acides aminés de la chitine,
• La Figure 31 : TM + novozyme : abondance relative des acides aminés de la chitine,
• La Figure 32 : TM + neutrase : abondance relative des acides aminés de la chitine,
• La Figure 33 : HI + prolyve : abondance relative des acides aminés de l'hydrolysat,
• La Figure 34 : GM + prolyve : abondance relative des acides aminés de l'hydrolysat,
• La Figure 35 : AD + prolyve : abondance relative des acides aminés de l'hydrolysat,
• La Figure 36 : Pureté gravimétrique de la chitine en fonction de différents procédés et différentes enzymes appliquées à T. molitor,
• La figure 37 : Pureté gravimétrique de la chitine en fonction de différents procédés et différents insectes avec la Prolyve,
• La Figure 38 : Pureté colorimétrique de la chitine en fonction de différents procédés et différentes enzymes avec T. molitor,
• La Figure 39 : Pureté colorimétrique de la chitine en fonction de différents procédés et différents insectes avec la Prolyve,
• La Figure 40 : Pureté par différence de la chitine,
• La Figure 41 : Degré d'acétylation de la chitine obtenue par le procédé pour T. molitor,
• La Figure 42 : Degré d'acétylation de la chitine obtenue par le procédé avec la Prolyve,
• La Figure 43 : Représentation de la cuticule par microscopie par fluorescence bi-photon,
• La Figure 44 : Degré de cristallinité des chitines obtenues, et
• La Figure 45 : Taille des protéines dans l'hydrolysat.

Other characteristics and advantages of the invention will appear in the examples which follow, given by way of illustration, with reference to the figures, which respectively represent:

• The Figure 1 is a diagram of a preferred embodiment of the method according to the invention,
• The Figure 2 a diagram comparing the degree of purity of the chitin obtained by an enzymatic process comprising one or more preliminary stages of grinding (s) and pressing,
• The Figure 3 is a diagram comparing the level of lipids measured in different fractions of the intermediate product from which the chitin has been extracted,
• The Figure 4 illustrates the distribution of lipids in the juice and the press cake obtained by an enzymatic process comprising preliminary stages of grinding and pressing or a preliminary stage of pressing,
• The Figure 5 is a diagram illustrating the distribution of proteins / peptides according to their molar masses (in%) in a hydrolyzate according to the invention and in the press juice,
• The Figure 6 is a diagram illustrating the relative distribution of the constituent amino acids of a hydrolyzate according to the invention,
• The Figure 7 is a diagram illustrating the relative abundance of amino acids in chitin;
• The Figure 8 : Effect of pressing on the ash content in the hydrolyzate - various insects,
• The Figure 9 : Effect of pressing on the ash content in the hydrolyzate - different enzymes,
• The Figure 10 : Ash content in the hydrolyzate with the grinding + pressing process - various insects,
• The Figure 11 : Relative abundance of large proteins according to different processes and different insects for hydrolysis performed with Prolyve,
• The Figure 12 : Relative abundance of large proteins as a function of different processes and different enzymes applied to T. molitor,
• The Figure 13 : Effect of pressing on the protein content of the hydrolyzate - different enzymes,
• The Figure 14 : Effect of pressing on the protein content of the hydrolyzate - different insects,
• The Figure 15 : Effect of pressing on the lipid content of the hydrolyzate - different enzymes,
• The Figure 16 : Effect of pressing on the lipid content of the hydrolyzate - different insects,
• The Figure 17 : Pepsi digestibility of the hydrolysates obtained - provisional,
• The Figure 18 : TM + prolyve: relative abundance of amino acids in the hydrolyzate,
• The Figure 19 : TM + sumizyme: relative abundance of amino acids in the hydrolyzate,
• The Figure 20 : TM + novozyme: relative abundance of amino acids in the hydrolyzate,
• The Figure 21 : TM + neutrase: relative abundance of amino acids in the hydrolyzate,
• The Figure 22 : HI + prolyve: relative abundance of amino acids in the hydrolyzate,
• The Figure 23 : GM + prolyve: relative abundance of amino acids in the hydrolyzate,
• The Figure 24 : AD + prolyve: relative abundance of amino acids in the hydrolyzate,
• The Figure 25 : Effect of pressing on the ash content in chitin - different enzymes,
• The Figure 26 : Ash content in the chitin of different insects,
• The Figure 27 : Lipid content in chitin,
• The Figure 28 : Amino acid level in chitin,
• The Figure 29 : TM + prolyve: relative abundance of amino acids of chitin,
• The Figure 30 : TM + sumizyme: relative abundance of amino acids of chitin,
• The Figure 31 : TM + novozyme: relative abundance of chitin amino acids,
• The Figure 32 : TM + neutrase: relative abundance of amino acids in chitin,
• The Figure 33 : HI + prolyve: relative abundance of amino acids in the hydrolyzate,
• The Figure 34 : GM + prolyve: relative abundance of amino acids in the hydrolyzate,
• The Figure 35 : AD + prolyve: relative abundance of amino acids in the hydrolyzate,
• The Figure 36 : Gravimetric purity of chitin as a function of different processes and different enzymes applied to T. molitor,
• The figure 37 : Gravimetric purity of chitin according to different processes and different insects with Prolyve,
• The Figure 38 : Colorimetric purity of chitin as a function of different processes and different enzymes with T. molitor,
• The Figure 39 : Colorimetric purity of chitin according to different processes and different insects with Prolyve,
• The Figure 40 : Purity by difference of chitin,
• The Figure 41 : Degree of acetylation of chitin obtained by the method for T. molitor,
• The Figure 42 : Degree of acetylation of chitin obtained by the process with Prolyve,
• The Figure 43 : Representation of the cuticle by bi-photon fluorescence microscopy,
• The Figure 44 : Degree of crystallinity of the chitins obtained, and
• The Figure 45 : Size of proteins in the hydrolyzate.

Example 1: Example of a process according to the invention

• Vitesse = 82 tr/min;
• W (énergie) = 3 HP (chevaux vapeur) soit 2,68 x 10⁶ J;
• Porosité (approximative) = 0,5 mm dans la première partie et 0,2 mm dans la dernière partie.

600 g of T. molitor larvae are introduced into a beaker, placed in a water bath at 100 ° C. and containing 600 mL of water, brought to the boil beforehand. After 5 minutes, the beaker is removed from the water bath, the larvae are drained, then mixed with a volume of water of 600 mL. The liquid thus obtained is pressed with an Angel type twin-screw press under the following conditions:

• Speed = 82 rpm;
• W (energy) = 3 HP (horsepower) or 2.68 x 10 ⁶ J;
• Porosity (approximate) = 0.5 mm in the first part and 0.2 mm in the last part.

• Surface de filtration de la cellule = 50 cm² ;
• Pression = 2 à 8 bars ;
• Température = 20 à 80 °C,
• Porosité = 25 à 80 µm ;
• Débit en fin de filtration = 100 à 250 mL/h.

A press juice is thus obtained and a press cake of 136.49 ± 4.48 g by wet weight, of which 100.22 ± 0.22 g are used in the following steps. All However, another type of press could have been used, insofar as this allows the extraction to obtain a press cake which is substantially similar in terms of water content and / or lipid content. By way of example, other tests were carried out with the Choquenet type filter press having the following characteristics:

• Filtration area of the cell = 50 cm ² ;
• Pressure = 2 to 8 bars;
• Temperature = 20 to 80 ° C,
• Porosity = 25 to 80 µm;
• Flow rate at the end of filtration = 100 to 250 mL / h.

The press cake is transferred into an Erlen Meyer containing 600 mL of a protease solution (Prolyve NP conc) at 1% (relative to the wet weight of the press cake), the whole is placed for 4 hours at 45 ° C with magnetic stirring (at a pH of about 6.5). The Erlen Meyer is then placed for 15 minutes in a water bath at 90 ° C. in order to deactivate the enzymes, the solution is then filtered at 0.45-0.5 μm while hot. The chitin thus obtained is dried for 24 hours at 70 ° C. 16.99 ± 1.77 g of chitin are thus obtained by this process. The hydrolyzate (filtrate obtained after hydrolysis) represents under these conditions 609.98 ± 10.9 g with a dry matter content of 5.05%, which, after lyophilization, gives 30.8 ± 0.55 g of l 'dry hydrolyzate.

Example 2: Influence of the mechanical steps prior to enzymatic hydrolysis on the degree of purity of the chitin obtained

Different types of mechanical pretreatment were tested, grinding (“grinding 1”) alone, grinding followed by pressing, grinding followed by pressing and a second grinding (“grinding 2”), as well as pressing alone.

For the pressing, the Angel-type press described in Example 1 was used.

1. Materials and methods Chitin production with grinding

200 g of T. molitor larvae are introduced into a beaker, placed in a water bath at 100 ° C. and containing 200 ml of water previously brought to the boil. After 5 minutes, the beaker is removed from the water bath, the larvae are drained, then mixed with a volume of water of 200 mL. The liquid thus obtained is transferred into an Erlen Meyer containing 2 g of protease (Prolyve), the whole is placed for 4 hours at 45 ° C with magnetic stirring (at a pH of about 6.5). The Erlen Meyer is then placed for 15 minutes in a water bath at 90 ° C. in order to deactivate the enzymes, the solution is then filtered at 0.45-0.5 μm while hot. The chitin thus obtained is dried for 24 hours at 70 ° C. 8.13 ± 0.27 g of chitin are thus obtained by this process.

Chitin production with grinding followed by pressing

200 g of T. molitor larvae are introduced into a beaker, placed in a water bath at 100 ° C. and containing 200 ml of water previously brought to the boil. After 5 minutes, the beaker is removed from the water bath, the larvae are drained, then mixed with a volume of water of 200 mL. The liquid thus obtained is passed through a twin-screw type press. 30 g of the press cake thus obtained are transferred into an Erlen Meyer containing 150 mL of water and 0.3 g of protease (Prolyve), the whole is placed for 4 hours at 45 ° C. with magnetic stirring (at a pH of approximately 6.5). The Erlen Meyer is then placed for 15 minutes in a water bath at 90 ° C. in order to deactivate the enzymes, the solution is then filtered at 0.45-0.5 μm while hot. The chitin thus obtained is dried for 24 hours at 70 ° C. 4.71 ± 0.11 g of chitin are thus obtained by this process.

Production of chitin with a first grinding ("grinding 1") followed by pressing and a second grinding ("grinding 2")

200 g of T. molitor larvae are introduced into a beaker, placed in a water bath at 100 ° C. and containing 200 ml of water previously brought to the boil. After 5 minutes, the beaker is removed from the water bath, the larvae are drained, then mixed with a volume of water of 200 mL. The liquid thus obtained is passed through a twin-screw type press. The press cake thus obtained is dried for 24 hours in an oven at 70 ° C., then ground to 250 μm. 10 g of the powder thus obtained are transferred into an Erlen Meyer containing 50 mL of water and 0.1 g of protease (Prolyve), the whole is placed for 4 hours at 45 ° C. with magnetic stirring (at a pH of 'about 6.5). The Erlen Meyer is then placed for 15 minutes in a water bath at 90 ° C. in order to deactivate the enzymes, the solution is then filtered at 0.45-0.5 μm while hot. The chitin thus obtained is dried for 24 hours at 70 ° C. 4.93 ± 0.12 g of chitin are thus obtained by this process.

Chitin production with pressing only

200 g of T. molitor larvae are introduced into a beaker, placed in a water bath at 100 ° C. and containing 200 ml of water previously brought to the boil. After 5 minutes, the beaker is removed from the water bath, the larvae are wrung out, then passed through a twin-screw press. 90 g of press cake thus obtained are transferred into an Erlen Meyer containing 450 mL of water and 0.9 g of protease (Prolyve), the whole is placed for 4 hours at 45 ° C. with magnetic stirring (at a pH of approximately 6.5). The Erlen Meyer is then placed for 15 minutes in a water bath at 90 ° C. in order to deactivate the enzymes, the solution is then filtered at 0.45-0.5 μm while hot. The chitin thus obtained is dried for 24 hours at 70 ° C. 6.48 ± 0.28 g of chitin are thus obtained by this process.

Chemical production of chitin

50 g of T. molitor larvae are introduced into a beaker, placed in a water bath at 100 ° C. and containing 50 ml of water, previously brought to the boil. After 5 minutes, the beaker is removed from the water bath, the larvae are drained, then mixed with a volume of water of 60 mL. The liquid thus obtained is transferred to a 1 L container and 500 ml of a 1M HCl solution are added. The whole is placed at 90 ° C. with stirring for 1 hour. The content is then filtered and the solid residue is transferred to a 1 L flask containing 500 mL of a 1M NaOH solution, the whole is placed at 90 ° C. with stirring for 24 hours. The residue is then filtered and placed in a ventilated oven at 70 ° C. for 24 hours. In this way 0.944 ± 0.005 g of chemically purified chitin is obtained.

Calculation of the degree of purity

The degree of purity of the chitin is determined by comparison of the mass of dry residue obtained relative to that resulting from a chemical extraction, approximately 5% of the initial dry matter.

Lipid level measurement

2 g of sample are placed in a beaker, 0.2 g of Na ₂ SO ₄ and 15 mL of CHCl ₃ / MeOH (2/1 v / v) are added thereto. The whole is placed under magnetic stirring for 20 minutes, then the solution is filtered, the residue is placed again in the beaker with 10 mL of CHCl ₃ / MeOH (2/1 v / v). The whole is placed under magnetic stirring for 15 minutes, then the solution is filtered, the solvent phases are combined and evaporated to constant weight. The lipid content is determined as a percentage of mass after extraction-evaporation relative to the initial mass of the sample (2 g).

2. Results

As can be seen on the Figure 2 , the process has an influence on the purity of the chitin obtained, the best results being obtained with a pressing at a minimum. The best result is obtained with grinding followed by pressing, namely a chitin exhibiting a degree of purity of 78% and the worst with grinding alone, namely a chitin exhibiting a degree of purity of 48%.

A more detailed analysis of the intermediate product from which the chitin was extracted, shows that a low level of lipids is favorable to a greater purity of the chitin obtained ( Figure 3 ). By “intermediate product” is meant the product entering enzymatic hydrolysis, that is to say resulting from the last step before hydrolysis, namely, according to the above production processes, the grinding step 1 or 2 or the pressing step.

Analysis of the level of lipids in chitin and hydrolysis juice makes it possible to note that depending on the level of initial lipids present in the intermediate product, the level of lipids in chitin is relatively stable, from 7 to 15%, while the level of lipids in the hydrolyzate varies from 11 to 47% ( Figure 3 ).

• la chitine qui résultera de l'hydrolyse ne sera pure qu'à 50% et comportera 10% lipides, et
• la teneur en lipides de l'hydrolysat avoisinera quant à elle les 40%.

More specifically, if the intermediate product has a fat content of 35%, then:

• the chitin which will result from the hydrolysis will be only 50% pure and will contain 10% lipids, and
• the lipid content of the hydrolyzate will be around 40%.

• la chitine obtenue après hydrolyse aura une pureté de 80% et aura une teneur en lipides de 8% et l'hydrolysat présentera également une faible teneur en lipides, de l'ordre de 10%.

On the other hand, if the lipid content of the intermediate product is 7%, then:

• the chitin obtained after hydrolysis will have a purity of 80% and will have a lipid content of 8% and the hydrolyzate will also have a low lipid content, of the order of 10%.

This indicates that when the level of the initial lipids is high, greater than 12%, the enzyme responsible for hydrolysis is caused to hydrolyze not only the proteins, but also the lipids by catalytic promiscuity. Thus, a similar level of lipids in chitin is obtained, namely 8.6 and 7.9% in cases where the initial lipids were 35 and 7% respectively. On the other hand, the purity of the chitin in this case increases from 48 to 84% respectively. Thus, out of the 52% impurities on the one hand and 16% on the other, 8% on average are due to lipids, the amount of proteins that have remained attached to the chitin is therefore 38 points higher if more lipids were present in the intermediate subjected to hydrolysis.

Finally, the importance of grinding upstream of pressing can also be studied ( Figure 4 ). It thus appears clearly that the distribution of lipids between the cake and the press juice is much more effective, 12.9 versus 87.1 versus 42.7 versus 57.3, when prior grinding has been carried out.

Example 3 : Analysis of the hydrolyzate

A detailed analysis of the hydrolyzate obtained in Example 1 was carried out.

1. Glucosamine content

The content of the hydrolyzate in glucosamines and certain other sugars was analyzed by gas chromatography after methanolysis and sillilation.

Procedure:

10 mg of the sample and 50 μg of internal standard are placed in 500 μL of a methanol / 3 N hydrochloric acid mixture for 4 hours at 110 ° C (or 24 hours at 130 ° C). The mixture is then neutralized with Ag ₂ CO ₃ . 50 μL of acetic anhydride are added in order to re-acetylate any osamines present. After one night in the dark and at room temperature, the samples are centrifuged (15 min, 3000 rpm) and the supernatant is evaporated. The compounds are then dissolved in 100 μL of pyridine and incubated overnight at room temperature with 100 μL of BSTFA (Supelco). The reagents are then evaporated and the residue taken up in 700 μL of CH ₂ Cl ₂ and injected by GC.

Temperature program: 1 minute at 120 ° C, ramp from 1.5 ° C / minute up to 180 ° C, then 2 ° C / minute up to 200 ° C.
Detection: FID
Column: HP-5MS (30m, 0.25mm internal diameter)
Internal standard: myo-inositol
The different contents were measured and calculated in two different ways (Table 2). It appears that the glucosamine is contained in the hydrolyzate in an amount of 0.1-0.15% by mass and with a ratio of 0.04-0.05 relative to the glucose. <b> Table 2: Distribution of glucosamine, mannose and glucose in the hydrolyzate </b> Methanolysis at 110 ° C Methanolysis at 130 ° C % mass Molar ratio % mass Molar ratio Glucose 1.6 ± 0.3 1 2 ± 0.4 1 Mannose 0.3 ± 0.05 0.15 ± 0.005 0.4 ± 0.08 0.15 ± 0.007 Glucosamine 0.1 ± 0.02 0.04 ± 0.007 0.15 ± 0.04 0.05 ± 0.003

2. Protein size

The size of the proteins / peptides of the hydrolyzate was evaluated by HPLC, Shimadzu 20A apparatus, at room temperature, on a Superdex Peptide 10/300 GL column, in 30% acetonitrile (ACN) buffer, trifluoroacetic acid (TFA) 0, 1%, with a flow rate of 0.4 mL / min. Detection was performed at 205 nm and the volume of sample injected was 20 µL.

Likewise, the protein / peptide size of the press juice prepared in Example 1 was also evaluated under identical conditions for comparison.

The comparison of the protein size between the press juice and the hydrolyzate ( Figure 5 ) clearly shows that the essential, 76%, of the peptides present in the hydrolyzate have a molar mass of between 130 and 900 Da. On the other hand, the part of the poorly digestible proteins (with a molar mass greater than 1300 Da) is reduced from 31% to 2%. Likewise, the proportion of peptides exhibiting a bitterness character (with a molar mass of less than 130 Da) goes from 38% to 15%. The digestibility and palatability criteria are therefore improved by the proposed enzymatic treatment.

3. Digestibility

The rate of pepsi digestibility of the hydrolyzate is estimated at 99.6% of the total proteins. It has been measured by an external laboratory, the method used complies with Directive 72/199 / EEC and was performed without degreasing.

4. Protein content

The protein level of the hydrolyzate is estimated at 84.8%. It was measured by an external laboratory, according to the Kjeldahl method with the correction coefficient of 6.25. The method used complies with EC regulation 152/2009.

5. Lipid content

The level of lipids in the hydrolyzate is estimated at 0.7 ± 0.5%. It was measured by an external laboratory using the so-called “hydrolysis” method adapted from EC regulation 152/2009 (method B).

6. Amino acid composition

The hydrolyzate obtained after treatment with the various enzymes was analyzed for its amino acid composition ( Figure 6 ). We observe the preponderance of proline, together with the presence of hydroxy proline (HYP) - metabolite of proline and amino acid absent in some organisms, for example crustaceans. However, proline and its metabolite, hydroxy proline, play an essential role in metabolism, in particular by allowing the synthesis of other amino acids such as arginine and glutamate. While most mammals are able to synthesize proline, the production of this amino acid by newborns, birds and fish is insufficient, which is why proline and hydroxy proline supplementation is sometimes used in order to '' increase the growth of certain animals. In addition, proline is the first amino acid contained in the milk of mammals, it is present there at 12%, on the other hand its content in plant proteins is much lower, only 2.9% in soybeans and 0 , 8% in corn. We therefore find in this hydrolyzate a proline content as high as in milk and much higher than that found in vegetable proteins.

The other amino acids present in large quantities are alanine, leucine and glutamate (with glutamine). Their relative amounts are greater than 9%. On the other hand, sulfur-containing amino acids such as cysteine and methionine are in small quantity, of the order of 0.5%.

The method used for determining these results (acid hydrolysis) did not allow the detection of amino acids such as tryptophan and arginine. In addition, asparagine has been completely transformed into aspartic acid and glutamine into glutamic acid and what is observed under the ASP and GLU peaks is in fact respectively the sum of aspartic acid and asparagine on the one hand. and glutamic acid and glutamine on the other hand, initially present in the hydrolyzate.

Example 4: Analysis of chitin

A detailed amino acid analysis of the chitin obtained in Example 1 was carried out.

The total amino acid content is 32.3 g per 100 g of copolymer (determined as the sum of the amino acids). The main amino acids present are valine, glycine, alanine and tyrosine ( Figure 7 ).

The analyzes were subcontracted and the results obtained according to the NF EN ISO 13904 method for tryptophan and NF EN ISO 13903 for the other amino acids.

Example 5 : Characterization of the hydrolyzate and of the chitin according to the production process implemented I. Materials and methods a) Material Insects

• un coléoptère : Tenebrio molitor (T. molitor ou TM),
• un lépidoptère : Galleria mellonella (G. melonella ou GM),
• un diptère : Hermetia illucens (H. illucens ou HI), et
• un orthoptère : Acheta domesticus (A. domesticus ou AD).

Different insects have been studied:

• a beetle: Tenebrio molitor ( T. molitor or TM),
• a lepidopteran: Galleria mellonella (G. melonella or GM),
• a Diptera: Hermetia illucens ( H. illucens or HI), and
• an orthopteran: Acheta domesticus ( A. domesticus or AD).

Enzymes

Different enzymes were used during the hydrolysis step.

• SAPU/g = une unité spectrophotométrique de protéase
• ΔA = absorbance corrélée
• i = ordonnée à l'origine
• 11 = volume final de réaction
• M = coefficient directeur de la courbe de calibration
• 30 = temps de réaction (en minutes)
• C = concentration de l'enzyme (g/mL) dans la solution enzymatique ajoutée
• 1 = 1 mL volume de la solution d'enzyme ajoutée

This measurement of the enzymatic activity is based on the principle of the measurement of tyrosine release at 275 nm during the hydrolysis of casein by a proteolytic enzyme (Valley research SAPU Assay method, by Karen PRATT). $$\frac{\mathit{IT\; STINKS}}{g}=\frac{\left(\mathrm{\Delta }\mathrm{AT}-i\right)\times 11}{m\times 30\times \mathrm{VS}\times 1}$$

• SAPU / g = one spectrophotometric unit of protease
• ΔA = correlated absorbance
• i = y-intercept
• 11 = final reaction volume
• M = director coefficient of the calibration curve
• 30 = reaction time (in minutes)
• C = concentration of enzyme (g / mL) in the added enzyme solution
• 1 = 1 mL volume of enzyme solution added

The calibration curve is obtained by measuring the absorbance of tyrosine solutions of different concentrations in a phosphate buffer (0.2 M, pH 7).

5 mL of a casein solution (0.7% m / v, 0.2 M phosphate buffer, pH 7, heated for 30 minutes at 90 ° C and added with 3.75 g / L _solution ) are incubated with 1 mL of the enzymatic solution (0.15 g in 100 mL of glycine buffer, 0.05M) to be tested at 37 ° C for 30 minutes. Then 5 mL of a TCA solution (18 g TCA, 11.35 g of anhydrous sodium acetate, 21 mL of glacial acetic acid, made up with demineralized water to 1 liter of solution) are added thereto, mixture on a vortex, filter and measure the absorbance at 275 nm.

The blank is prepared identically but without adding enzyme, 1 ml of demineralized water is added instead in order to find the same reaction volume.

The activities thus measured for the various enzymes used (Prolyve NP, Novozyme 37071, Neutrase and Sumizyne) are listed in Table 3.

b) Production processes Production process with grinding alone (referred to as "grinding" in the figures)

600 g of fresh insects (whether larvae in the case of T. molitor, G. melonella or H. illucens, or locusts in the case of A. domesticus) are introduced into an enclosure where they are slaughtered with steam (115 ° C, 5 minutes). The insects are then introduced into a mixing device and 75 mL of water per 100 g of insects are added, the whole is then mixed. 100 g (wet weight) of product thus obtained are then introduced into a three-necked flask fitted with a condenser and a mechanical stirrer, a proteolytic enzyme with an activity equivalent to 3789 SAPU is then added. The reaction is then brought to 45 ° C. for 4 hours. The temperature is then brought to 90 ° C. for 15 minutes, the reaction mixture is finally filtered (0.40 - 0.45 μm). The residue is dried for 24 hours at 70 ° C.: it is therefore the chitin obtained by the enzymatic purification route; the filtrate is frozen and is lyophilized: it is therefore the hydrolyzate.

The process is identical regardless of the insect or enzyme studied.

Production process with grinding followed by pressing (referred to as "grinding + pressing" in the figures)

600 g of fresh insects (whether larvae in the case of T. molitor, G. melonella or H. illucens, or locusts in the case of A. domesticus ) are introduced into an enclosure where they are slaughtered with steam (115 ° C, 5 minutes). The insects are then introduced into a mixing device and 75 mL of water per 100 g of insects are added, the whole is then mixed and pressed (twin-screw press, or filter press, or other pressing system). 100 g (wet weight) of product thus obtained are then introduced into a three-necked flask fitted with a condenser and a magnetic stirrer, 500 ml of water are added as well as a proteolytic enzyme with an activity equivalent to 3789 SAPU . The reaction is then brought to 45 ° C. for 4 hours. The temperature is then brought to 90 ° C. for 15 minutes, the reaction mixture is finally filtered (0.40 - 0.45 μm). The residue is dried for 24 hours at 70 ° C.: it is therefore the chitin obtained by the enzymatic purification route; the filtrate is frozen and is lyophilized: it is therefore the hydrolyzate.

The process is identical regardless of the insect or enzyme studied.

c) Analyzes Ash content measurement

The ash content was determined using the method governed by EC regulation 152/2009 of 27-01-2009.

Protein size measurement

100 mg of sample are placed in 10 mL of phosphate / NaCl buffer (pH 7.4, 0.137 mM). The sample was stirred for one minute (vortex), centrifuged at 900 g for 1 min and then filtered through a 0.45 μm membrane. The analysis was carried out on a steric exclusion chromatography system, with the Nucleogel GFC-300 column, the eluent used is phosphate / NaCl buffer (pH 7.4, 0.137 mM), the flow rate is 1.0 mL / min, the detection is carried out by a UV detector at 280 nm.

Protein content measurement

The protein level is obtained by the Dumas method, with the conversion coefficient of 6.25, adapted from standard NF EN ISO 16634-1

Measurement of lipid content

The lipid content is obtained by a method adapted from EC regulation 152/2009 - process B - SN.

Pepsic digestibility

Peptic digestibility is measured by the method described in Directive 72/199 / EC.

Relative abundance of amino acids

The abundance of amino acids was determined by a method resulting from regulation EC 152/2009 of 27-01-2009 - SN. The tryptophan content was determined separately by a method adapted from EC regulation 152/2009 of 27-01-2009 - SN. Relative abundance was calculated by relating the content of each amino acid to the ratio of amino acids.

Amino acid level

Amino acid level was determined by summing up the individual values obtained for each amino acid, including tryptophan.

Gravimetric purity

The gravimetric purity is determined by comparison of the dry residue mass obtained relative to that resulting from a chemical extraction, the latter being evaluated at about 5% of the initial dry matter.

Colorimetric purity

The color of the sample was estimated thanks to the analysis of photographs of samples using the ImageJ software according to the three colors red, green and blue (RGB or RGB in English), their average representing a rating of the real color. A sample of shrimp chitin marketed by Chitine France was taken as a standard (100% purity) and the colorimetric purity of the samples produced was calculated as a percentage of this color (color ratio of the sample / color of the standard) .

Purity by difference

In the case of this measurement, the amounts of known impurities (amino acids, lipids and ash) were subtracted from the absolute purity value (100%) to obtain the purity value estimated by difference; ie a sample which contains 30% proteins, 10% lipids and 1% ash, is therefore assigned a purity of 100-30-10-1 = 59%

Degree of crystallinity

The measurements were carried out according to the WAXS technique (wide angle X-ray scattering) on the Bruker D8 Advance device (A25 DaVinci design) equipped with a Lynxeye XE detector. The results were interpreted according to the method described in Loelovich, M. Res. Rev .: J. Chem. 2014, 3, 7-14 .

Degree of acetylation

The measurements were carried out using a ¹³ C CP / MAS NMR apparatus, Bruker brand and equipped with an 800 MHz magnet.

The analysis of the results was carried out in accordance with that described in Simionatto Guinesi, L .; Gomes Cavalheiro, ET Thermochimica Acta 2006, 444, 128-133 and Heux, L .; Brugnerotto, J .; Desbrières, J .; Versali, M.-F .; Rinaudo, M. Biomacromolecules 2000, 1, 746-751 .

Measurement of atomic and functional abundance at the surface

The measurements were carried out using an XPS Escolab 250 device, brand VG Scientific and equipped with an RX Ka A1 source (1486.6 eV), a monochromator and a magnetic lens.

The samples, powdered beforehand, were placed under vacuum for 48 to 72 hours and then analyzed.

Representation of the cuticle by bi-photon fluorescence microscopy

Multiphoton microscopy on DISCO devices (Synchrotron Soleil).

Lambda exc = 810nm, power 18%, turquoise blue and ocher filter 406/15 (SHG), blue filter 460/60, green filter 550/88.

II. Hydrolyzate a) Ashes

The effect of pressing is very significant on the ash content in the hydrolyzate obtained, whatever the insect studied ( Figure 8 ). Indeed, the decrease in ash content can reach 52%, the declining proportion is relatively similar when it comes to insects naturally rich or not in minerals. Thus, in the case of H. illucens - the ash content goes from 7.5 g / 100 g of dry matter when the process with grinding is used to 3.8 g / 100 g of dry matter, when one adds a pressing step, ie . 49% decrease; in the case of T. molitor, we thus go from 5 g / 100 g to 2.4 g / 100 g, ie 52% reduction.

The effect of pressing is also significant on the level of ash in the hydrolyzate obtained, whatever the enzyme used ( Figure 9 ). In particular, it is noted that T. molitor, whatever the enzyme, the level of ash in the hydrolyzate is less than 3% when the method with pressing is used.

The level of ash in the hydrolyzate (obtained by the crushing + pressing process) is less than 4 g / 100 g of dry matter, regardless of the insect studied ( Figure 10 ).

b) Size of proteins in the hydrolyzate

The use of the pressing step clearly improves the performance of proteolytic enzymes, and those regardless of the insect ( Figure 11 ) or the enzyme used ( Figure 12 ). The relative abundance of large proteins thus drops significantly compared to the process comprising only the grinding step, the final hydrolyzate comprising only 10.3% maximum in the case of the hydrolysis carried out with the prolyve, whatever the insect; and no more than 17.5% in the case of the hydrolysis of T. molitor, whatever the enzyme used.

c) Protein content

The protein content of the hydrolyzate depends very strongly on the process used. Thus when the grinding is applied alone, the proteins represent only 53.59 ± 1.5% of the dry matter of the hydrolyzate of T. molitor thus obtained, and this whatever the enzyme used ( Figure 13 ). On the other hand, when grinding is followed by pressing, the level of proteins in the hydrolyzate increases to 84.58 ± 1.4%, it thus undergoes an increase of 53 - 62%, depending on the enzyme used.

This increase is all the greater when the insect was initially poor in proteins, thus in the case of G. melonella, the protein rate goes from 41.25% to 71%, undergoing an increase of almost 86% and in the case of H. illucens, the increase is even 118%, since we go from 34, 2% to 74.5% ( Figure 14 ).

d) Lipid content

Pressing has a major influence on the lipid content of the hydrolyzate, whatever the enzyme ( Figure 15 ) or the studied insect ( Figure 16 ). In fact, the lipid content drops drastically from 51.4-97.7%, thus dropping from 28.6 to 13.9% in the case of H. illucens (51.4% decrease) and from 33.85 to 0.9% in the case of T. molitor with novozyme (97.3% decrease).

e) Peptic digestibility

The pepsi digestibility of the hydrolysates thus obtained is very high, greater than 96% whatever the enzyme or the insect studied ( Figure 17 ).

f) Amino acid abundance

The use of the pressing step allows a better extraction of amino acids more present at the level of the cuticle, such as alanine and tyrosine and, to a lesser extent, valine, serine and glycine, and this whatever the enzyme or the insect studied ( Figures 18 - 24 ). These results are also to be compared with those concerning the amino acids present at the level of enzymatically purified chitin ( Figure 29-35 ).

Note: if the relative abundance of certain amino acids, including aspartic acid and glutamate, in particular, decreases, their amounts remain the same, or even increase slightly in certain cases, since the total amount of amino acids extracted by the process with a pressing step increases (see protein rate in the hydrolyzate)

III. Chitin a) Ashes

The level of ash in chitin is also affected by the pressing step, although to a lesser extent than for the hydrolyzate. A dimuniton ranging from 25% to 28.6% is thus observed depending on the enzyme used ( Figure 25 ).

The ash content in chitin (obtained by the crushing + pressing process) is extremely low, whatever the insect ( Figure 26 ) studied. Thus, in the case of T. molitor, the maximum ash content is 1.05 g / 100 g of dry matter, and even for insects naturally rich in minerals it remains below 3.5 g / 100 g of matter. dried.

b) Lipid content in chitin

Although to a slightly lesser extent than for the hydrolyzate, the lipid content in chitin also decreases when the pressing step is added to the process ( Figure 27 ). The efficiency depends mainly on the enzyme, 15% for sumizyme and up to 60% for novozyme. Regardless of the insect, this reduction is between 50% in the case of H. illucens and 58% in the case of T. molitor, when the reaction is carried out with the prolyve.

For all insects, the lipid content is less than 12%, and for non-flying insects it is even ≤6%.

c) Relative rate and abundance of amino acids in chitin

The pressing step allows the removal of a greater part of the proteins attached to the chitin. The direct measurement of the protein level being made difficult given the amide function present at the level of the very structure of chitin, we approached this protein level by the sum of amino acids ( Figure 28 ). Thus, we can see that the sum of amino acids decreases, from 5 to 54%, when the pressing step is added to the process, regardless of the enzyme or the insect studied.

The relative abundance of amino acids is weakly affected by the pressing step, some amino acids nevertheless, including alanine, seem to be better extracted when the pressing step is performed ( Figures 29 to 35 ). Nevertheless we can see that for all insects, alanine, tyrosine and proline, as well as, to a lesser extent, valine, glycine, leucine and serine are amino acids mainly attached to chitin , their rate is on average between 23 - 40% of all amino acids, and it is also among these amino acids that we note the most significant decreases in chitin and the most significant increases (we arrive at 7-30% maximum) at the hydrolyzate level ( Figures 18-24 ) when adding a press step in the process.

d) Gravimetric purity

Regardless of the enzyme ( Figure 36 ) or the insect ( Figure 37 ) studied, the gravimetric purity is improved by the use of the pressing step. It thus goes from 48.7% to 77.1% in the case of T. molitor with the prolyve and from 33.7% to 87.9% in the case of H. illucens with the prolyve.

e) Colorimetric purity

An improvement, admittedly less clear compared to the gravimetric purity, of the colorimetric purity is also observed when the pressing step is part of the process, and this whatever the enzyme or the studied insect ( Figures 38 and 39 ).

f) Purity by difference

Due to the decrease in the level of lipids, ash and amino acids in the chitin obtained after the process with pressing, the purity by difference of this chitin, increases significantly, by 13% in the case of H. illucens and up to 74% in that of T. molitor with prolyve, and this whatever the insect or the enzyme studied ( Figure 40 ).

g) Degree of acetylation of chitin

The degree of acetylation is significantly affected by the addition of the pressing step in the process ( Figures 41 and 42 ). Indeed, the elimination of lipids and amino acids from hemolymph allows greater accessiility of enzymes to the surface of the cuticle, which contributes to improving the cleavage of peptide bonds of proteins linked to chitin, but also, by catalytic promiscuity, cleavage of the amide links of chtin. Thus the degree of acetylation of the chitin of T. molitor resulting from the process is between 76 and 79%, whereas in the absence of the pressing step, it is at 91% and even chemical hydrolysis, carried out under drastic temperature and reagent conditions results in a degree of acetylation of 85%.

For the other insects considered, in particular G. melonella and A. domesticus, the degree of acetylation is also affected by the process, so it goes from 83 to 74% in one case and from 100 to 95% in the other, respectively.

The solubility and the processability of chitin being particularly linked to its degree of acetylation, this concomitant hydrolysis of the amide bond of the N -acetyl-glucosamine units of chitin is an unexpected positive effect of the enzymatic hydrolysis carried out in these conditions.

h) Atomic and functional abundances at the surface

The distribution of atoms on the surface of the chitin as the purification progresses shows an increase in the relative rate of oxygen and a decrease in the relative rate of carbon (Table 4).

Likewise, the bonds of carbon atoms at the surface tend to go from bonds predominantly of the hydrocarbon type (C-H, C-C), to bonds of the alcohol (C-O) or carbonyl (C = O) type. Amide-type bonds (N-C = O) also decrease.

What, however, seems the most representative is the alcohol bond / hydrocarbon bond (CO / CH) ratio, in the context of enzymatically purified chitin this ratio is between 0.31 and 0.56, but more specifically between 0.39 and 0.41.

• « cuticule brute », on vise la chitine de la cuticule à l'état natif, analysée directement après arrachage à l'insecte par dissection ;
• « chitine purifiée enzymatiquement (novozyme) », on vise une chitine issue d'un procédé comportant un broyage + pressage et une hydrolyse enzymatique en présence de l'enzyme Novozyme 37071 ;
• « chitine purifiée enzymatiquement (prolyve) », on vise une chitine issue d'un procédé comportant un broyage + pressage et une hydrolyse enzymatique en présence de l'enzyme Prolyve NP ;
• « chitine purifiée enzymatiquement », on vise une chitine issue d'un procédé comportant un broyage + pressage et une hydrolyse enzymatique en présence de l'enzyme prolyve ;
• « chitine pure », on vise une chitine obtenue par purification chimique, identique à celle utilisée pour la détermination de la quantité de la chitine dans l'insecte
  

In this Table 4, by:

• “Raw cuticle” means the chitin of the cuticle in the native state, analyzed directly after the insect has been torn off by dissection;
• “Enzymatically purified chitin (novozyme)” is intended for a chitin resulting from a process comprising grinding + pressing and enzymatic hydrolysis in the presence of the enzyme Novozyme 37071;
• "Enzymatically purified chitin (prolyve)" is intended a chitin resulting from a process comprising a grinding + pressing and an enzymatic hydrolysis in the presence of the enzyme Prolyve NP;
• “Enzymatically purified chitin” is intended for a chitin resulting from a process comprising grinding + pressing and enzymatic hydrolysis in the presence of the prolyve enzyme;
• "Pure chitin" means a chitin obtained by chemical purification, identical to that used for determining the amount of chitin in the insect
  

i) Representation of the cuticle by bi-photon fluorescence microscopy

The partial purification of chitin by the enzymatic route according to the process according to the invention allows the flexibility of the assembly to be maintained by the preservation of protein "filling" of the chitin structures, compared to chemical purification, while removing the protein overload. outside the structure (see Figure 43 ).

The terminology used on Figure 43 is the same as that defined in point III h) above.

j) Degree of crystallinity

Whatever the insect or the enzyme studied, the degree of crystallinity, ie the ratio of crystalline and amorphous areas, of the chitin obtained is between 0.42 and 0.61.

Generally, in the literature, we speak of the crystallinity index, i.e. the ratio of peak heights (and not of areas). This measurement carries a significant risk of error. Nevertheless, and for the purpose of comparison, the crystallinity index of the samples was also measured, and it is between 88 and 95% for all insects, and is even greater than 90% for beetles and lepidoptera.

Example 6 : Process according to the invention with recovery of the protein fraction obtained from the press juice I. Materials and methods a) Material Insect

• un coléoptère : Tenebrio molitor (T. molitor ou TM).

The following insect has been studied:

• a beetle: Tenebrio molitor ( T. molitor or TM).

Enzyme

The Prolyve was used during the hydrolysis.

b) Production processes Production process with grinding followed by pressing (referred to as "grinding + pressing" in the figures)

Several batches were processed as follows: 600 g of fresh T. molitor larvae are introduced into an enclosure where they are slaughtered with steam (115 ° C., 5 minutes). The insects are then introduced into a mixing device and 75 mL of water per 100 g of insects are added, the whole is then mixed and pressed (twin-screw press, or filter press, or other pressing system).

The press cake is then dried, 70 ° C. overnight. The press juice, on the other hand, is centrifuged for 30 minutes with a force of 3000 g, and the solid part is collected - insoluble proteins of the hemolymph.

125 g of press cake and 125 g (wet weight, approximately 30% dry weight) of insoluble hemolymph proteins are then introduced into a three-necked flask fitted with a condenser and a magnetic stirrer, 1250 mL of water are added as well as a proteolytic enzyme with an activity equivalent to 9500 SAPU. The reaction is then brought to 45 ° C. for 4 hours. The temperature is then brought to 90 ° C. for 15 minutes, the reaction mixture is finally filtered (0.40 - 0.45 μm). The residue is dried for 24 hours at 70 ° C.: it is therefore the chitin obtained by the enzymatic purification route; the filtrate is frozen and is lyophilized: it is therefore the hydrolyzate.

c) Analyzes

The measurement of the ash content, the measurement of the lipid content, the relative abundance of amino acids and the measurement of the size of the proteins were carried out as in Example 5.

II. Characterization of the products obtained

The products obtained, the chitin and the hydrolyzate, were analyzed and characterized (Table 5, Figure 45 ). <b> Table 5 </b>: Characterization hydrolyzate Chitin Ashes 4.1 0.5 Lipids 12.85 3.7 digestibility 95 n / A Amino acids (relative abundance (%) ASP 9.7 5.4 GLUE 12.8 6.7 TO THE 9.0 13.7 ARG 2.5 3.6 CYS 1.1 0.5 GLY 5.9 9.4 HIS 3.3 3.9 ISLE 5.4 4.8 LEU 8.5 8.0 Lilies 6.5 2.9 MET 1.5 0.4 PHE 4.0 2.6 PRO 6.9 8.3 SER 4.4 5.8 THR 4.9 3.5 TYR 4.7 11.3 VAL 7.7 9.0 TRP 1.3 0.6 na not applicable

Claims (14)

1. Hydrolysate prepared from insects comprising at least 40% by weight of proteins based on the total weight of dry matter, an ash content of less than 3% by weight based on the total weight of dry matter, and a content of watersoluble proteins having a size larger than 12,400 g/mol of less than 50%.
2. Method for the production of at least one product of interest from insects, comprising the following steps:

   (i) grinding the insect cuticles,

   (ii) pressing the insect cuticles, and then

   (iii) enzymatic hydrolysis of the insect cuticles with a proteolytic enzyme.
3. Method according to claim 2, comprising a step of killing the insects prior to the grinding step.
4. Method according to claim 2 or 3, further comprising a step of treatment of the insect cuticles with an oxidizing agent prior to enzymatic hydrolysis.
5. Method according to any one of claims 2 to 4, in which the insect or insects is/are selected from the group constituted by the Coleoptera, the Lepidoptera, the Orthoptera and the Diptera.
6. Method according to any one of claims 2 to 5, in which the protease is selected from the group constituted by aminopeptidases, metallocarboxypeptidases, serine endopeptidases, cysteine endopeptidases, aspartic endopeptidases, metalloendopeptidases.
7. Method according to any one of claims 2 to 6, in which the product of interest is chitin and/or chitosan.
8. Method according to any one of claims 2 to 6, in which the product of interest is a hydrolysate.
9. Method for the production of chitin from insect cuticles, comprising the following steps:

   a) killing the insects,

   b) grinding the insects,

   c) pressing the insects,

   d) enzymatic hydrolysis of the insect cuticles with a proteolytic enzyme,

   e) recovery of the chitin,
   the insect cuticles optionally being able to be treated with an oxidizing agent before step d).
10. Chitin obtainable by a method according to any one of claims 2 to 7, and 9.
11. Method for the production of a hydrolysate from insects, comprising the following steps:

    a) killing the insects,

    b) grinding the insects,

    c) pressing the insects,

    d) enzymatic hydrolysis of the insect cuticles with a proteolytic enzyme,

    e) recovery of the hydrolysate,
    the insect cuticles optionally being able to be treated with an oxidizing agent before step d).
12. Hydrolysate obtainable by a method according to any one of claims 2 to 6, 8, and 11.
13. Method for the production of chitosan from insect cuticles, comprising the following steps:

    a) killing the insects,

    b) grinding the insects,

    c) pressing the insects,

    d) enzymatic hydrolysis of the insect cuticles with a protease,

    e) recovery of the chitin,

    f) deacetylation of the chitin recovered,

    g) recovery of the chitosan,
    the insect cuticles optionally being able to be treated with an oxidizing agent before step d).
14. Chitin comprising an amino acid content less than or equal to 45% by weight based on the total weight of dry matter, an ash content less than or equal to 2.5% by weight based on the total weight of dry matter, a purity by difference greater than or equal to 45%, a degree of acetylation greater than or equal to 70% and a degree of crystallinity greater than or equal to 0.42.

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